The present invention relates to a method of anti-coagulating blood, blood plasma or synovial fluid products with moderately calcium binding anti-coagulants, and optionally adjusting the calcium ion activity up to physiological levels with a soluble calcium salt.
Biological fluids, in particular blood, lymph, synovial fluid and cerebral liquor and cell free preparations of these, e.g. blood plasma, can undergo drastic changes in rheological, i.e. visco-elastic, properties. The fluids can change from a liquid having rheological properties similar to water, to a gel having rheological properties more like cottage cheese. The described rheological transition, commonly denoted coagulation or clotting, can occur when the biological fluid is removed from its natural environment and is collected in some man-made container.
To prevent coagulation upon collection of biological fluid, some substance with coagulation-inhibiting properties may need to be added. Substances used to inhibit coagulation, also called anti-coagulants, include oxalate, EDTA, citrate, heparin and hirudin. These anti-coagulants either cause specific inhibition of enzymes involved in coagulation or modify conditions within the biological fluid so that the coagulative processes are unable to proceed. These conditions include free calcium ion concentration, also called Ca2+ activity, and pH.
According to prior art theory, the mechanism of action for some of the anti-coagulants is to form complexes with Ca2+ ions and thereby reduce the levels of free Ca2+, i.e. reduce the Ca2+ activity, in a biological fluid to non-effective levels. It is thereby understood that the Ca2+ activity is required at a certain level for the coagulation processes to proceed. The present invention pertains particularly to anti-coagulants of this later kind when coagulation is hampered when Ca2+ activity is below 0.8 mM or the pH is below 7.
If the biological fluid is anti-coagulated by reduction of the Ca2+ activity, then the biological fluid can be brought back to a coagulable state by the addition of Ca2+, e.g. in the form of a CaCl2 solution, to restore the Ca2+ activity to coagulation-permitting levels.
The procedure of anti-coagulating a biological fluid in a reversible manner, i.e. in a manner that allows a return of the fluid to a coagulation-permitting state, is common, and necessary, for analysis of the coagulation activity of the fluid. Oxalate, EDTA and citrate are examples of anti-coagulants that, according to prior art, function by reducing the Ca2+ activity in the biological fluid. Further, according to prior art, any substance that sufficiently reduces Ca2+ activity of a biological fluid will function as an anti-coagulant.
According to prior art, all substances that function as anti-coagulants by reduction of the Ca2+ activity form relatively stabile Ca2+ complexes which are characterized by apparent dissociation constants of 0.5 mM or less. Citrate, for example, which together with EDTA is the most commonly used anticoagulant of this kind, forms Ca2+ complexes with an apparent dissociation constant at physiological conditions of about 0.4 mM. The Ca2+ EDTA complexes and Ca2+ oxalate complexes are considerably tighter.
As discussed above, analysis of coagulation analytes in biological fluids are commonly performed on citrate anti-coagulated samples. By virtue of its relatively low affinity for Ca2+ and by use of minimal amounts, anti-coagulation with citrate is considered to be gentle with regard to causing denaturation and destabilization of proteins and lipid-protein structures of the biological fluid. Still, it may be assumed, that citrate anti-coagulation has some adverse effects. Factor VIII, for example, is less stabile in citrated blood and blood plasma than in the circulation. Also the results of activated prothrombin time (APTT) analysis of citrated blood or blood plasma displays limited stability. Thus, according to prior art, some coagulatory analytes display a degree of instability that necessitates special considerations in clinical laboratory routines. These more rigorous routines add costs and inconvenience to medical laboratory procedures.
When analyzing an anti-coagulated sample of a biological fluid for various coagulatory activities, the sample is mixed with reagents which include Ca2+. The amount of added Ca2+ is sufficient to give the reaction mixture a Ca2+ in the range of 0.8 to 8 mM at which conditions the coagulatory activities are displayed.
Although commonly used, the procedure of first anti-coagulating a sample of biological fluid by reduction of Ca2+ activity is undesirable in some respects. For example, denaturation and destabilization of the structures of interest as discussed above. Further, it is impossible, in a given procedure, to exactly balance out the effects of added anti-coagulant by addition of Ca2+. The Ca2+ of the recalcified sample is likely to be different from the native fluid, and in a series of samples of such a fluid, the variation in Ca2+ activity is likely to be greater than the variation in the original native fluid. This deviation from the native levels, also denoted physiological levels, of Ca2+ activity displayed in recalcified biological fluids most likely has undesirable influence on coagulation assay systems and their results.
A related medical field in which anti-coagulation by reduction of Ca2+ causes practical concerns is blood banking. Blood or blood plasma that is to be used in transfusion is commonly anti-coagulated. This is because, with present techniques, native blood and blood plasma can only be handled for a short time period outside the organism without coagulating.
Because of low toxicity, rapid metabolization and relatively good anti-coagulant properties, citrate is the commonly used anticoagulant in blood banking. In many cases, the citrate content in transfused blood or blood plasma does not cause adverse effects for the receiving patient. But in some cases, especially in patients with liver dysfunction, citrate anti-coagulated blood and blood plasma are not well tolerated. Due to slow metabolism of citrate in these patients, the citrate causes a notable reduction in Ca2+ activity which results in cramps and gastrointestinal discomfort.
There is a need for an improved method of anti-coagulating blood, blood plasma and synovial fluid products to improve the properties of the products for such applications as transport and storage of the products, use of the products for transfusion purposes and analysis of hemostatic or coagulative properties of the products.
The present invention provides a method of anti-coagulating a blood, blood plasma or synovial fluid product. The method comprises addition of iso-citrate and optionally a soluble calcium salt.
The method of the invention is for example useful for accomplishing 1) increased stability and reduced denaturation of coagulation analytes in the anti-coagulated product, 2) closer to physiological levels of Ca2+ activity and decreased variation in Ca2+ activity of assay systems for coagulation analytes, and 3) reduction in the Ca2+ lowering effect when citrate anti-coagulated blood or blood plasma is transfused into patients.
Thus, one aspect of the invention is directed to a method of anti-coagulating a blood, blood plasma or synovial fluid product comprising the step of adding to the product an effective amount of iso-citrate.
In an embodiment of this aspect of the invention the method additionally comprises the step of adjusting the calcium ion activity of the anti-coagulated product up to physiological levels with a soluble calcium salt.
In this context it can be mentioned that the calcium ion activity of the anti-coagulated product of the invention can be reduced by the addition of citrate or other calcium binding substance and that the pH can be adjusted for specific purposes.
In a preferred embodiment of this aspect of the invention the effective amount of iso-citrate is 5-50 mmol per liter of blood product or 7-70 mmol per liter of blood plasma or synovial fluid product, and in a most preferred embodiment the amount of iso-citrate is 15-25 mmol per liter of blood product or 2-35 mmol per liter of blood plasma or synovial fluid product.
Another aspect of the invention is directed to a blood, blood plasma or synovial fluid product containing an anti-coagulating amount of anti-coagulant comprising iso-citrate. In an embodiment of this aspect of the invention the anti-coagulant comprises citrate, in addition to iso-citrate.
In a preferred embodiment of this aspect of the invention the anti-coagulating amount of iso-citrate is 5-50 mmol per liter of blood product or 7-70 mmol per liter of blood plasma or synovial fluid product, and in a most preferred embodiment anti-coagulating amount of iso-citrate is 15-25 mmol per liter of blood product or 25-35 mmol per liter of blood plasma or synovial fluid product.
A further aspect of the invention is directed to the use of a blood, blood plasma or synovial fluid product according to the invention for transport and storage of the product. The transported and stored blood, blood plasma or synnovial fluid product may then be used directly for transfusion into patients.
Yet another aspect of the invention is directed to the use of a blood, blood plasma or synovial fluid product according to the invention for analysis of hemostatic or coagulative properties of the product, i.e. for diagnostic purposes.
Still another aspect of the invention is directed to a blood sampling container comprising an isotonic or slightly hypertonic solution of iso-citrate.
Another embodiment of a blood sampling container of the invention comprises a solution of 0.1-0.5 M iso-citrate in one tenth of the container volume.
The blood sampling containers according to the invention may additionally comprise, together with the iso-citrate, an effective amount of calcium chloride resulting in a calcium ion activity of up to approximately 1 mM.
The blood sampling containers of the invention are preferably conventional, optionally evacuated, test tubes, syringes or plastic bags used in the handling of blood and blood plasma products, and are ready for delivery to laboratories responsible for blood sampling and analysis.
Yet another aspect of the invention is directed to reagents for analysis of blood, blood plasma or synovial fluid products comprising iso-citrate. The reagents are provided in containers with labels and instructions for use.